Biotechnological methods are used to an increasing extent in the production of proteins, peptides, nucleic acids and other biological compounds, for research purposes as well as in order to prepare novel kinds of drugs. Due to its versatility and sensitivity to the compounds, chromatography is often the preferred purification method in this context. The term chromatography embraces a family of closely related separation methods, which are all based on the principle that two mutually immiscible phases are brought into contact. More specifically, the target compound is introduced into a mobile phase, which is contacted with a stationary phase. The target compound will then undergo a series of interactions between the stationary and mobile phases as it is being carried through the system by the mobile phase. The interactions exploit differences in the physical or chemical properties of the components in the sample.
In liquid chromatography, the target compound is present in a liquid together with one or more contaminants or undesired substances. Said liquid is contacted with a stationary phase, known as a matrix, which is commonly comprised of either a collection of homogenous, porous or non-porous particles or a monolith of organic or inorganic origin. The properties of the separation matrix will in large decide the efficiency obtained when used in a separation process, such as chromatography. Usually, a separation matrix is comprised of a support to which groups capable of interaction with the target and known as ligands have been coupled. Thus, the ligands will impart to the supports the ability to effect the separation, identification, and/or purification of molecules of interest. In the prior art, a number of different techniques for controlling the density of ligands on a support have been suggested, which techniques generally fall into one of the following four categories:    a) Manipulation of reaction conditions which activate the matrix, i.e. which introduce a reactive group which can couple the ligand. This often involves varying the concentration of activating reagents, reaction time, reaction temperature, pH, or combinations of these variables. Thus, the efficiency of the reaction, i.e. the extent of desired reaction as opposed to competing side reactions, will be strongly influenced by reaction conditions.    b) Manipulation of reaction conditions during the actual coupling of the ligand to the support. This may involve varying the concentration and/or the total amount of ligand the support is challenged with, ionic strength of the coupling buffer, and type of salt in the coupling buffer as well as the variables of time, temperature, pH, etc., mentioned above. Similarly to the technology described above, due to the strong influence of the reaction conditions, this method may also prove difficult to apply in a practical and reproducible manner.    c) Manipulation of the amount of reactive or activatable groups incorporated into the support by varying composition at the time of its formation. For a polymeric support, this would include varying the nature and/or amount of monomer during the polymerisation. Obviously, one should subsequently apply the techniques of a and/or b above in a second step to couple the ligand.    d) For polymeric ligands, manipulation of the amount of ligand incorporated into the polymer by preparation of a polymerisable ligand monomer and varying the concentration of this monomer in the monomer feed during polymerisation. A drawback with this technology is that many ligands useful for chromatographic separations contain functional groups which are incompatible with the conditions necessary for formation of the desired polymer, such as by being unstable under the contemplated polymerisation conditions or by interfering with the polymerisation reaction.
A different method of controlling the density of ligands on a support was suggested in U.S. Pat. No. 5,561,097 (Gleason et al.), which relates to a method of providing an optimised ligand density on a polymeric support, which method is stated to be obtained in a practical and reproducible manner. This can be achieved by a method comprising a step of reacting ligand and a quencher molecule with activated sites on an azlactone functional support under conditions that promote competition of ligand with quencher for the activated sites. One advantage presented is that the method is a single step procedure, without any need of a separate step to activate or deactivate reaction sites on the support. The method disclosed is stated to be especially advantageous for the coupling of small molecules. A disadvantage of this method is that in order not to favour ligand over quencher, an understanding of the reaction kinetics for ligand and reaction kinetics for quencher will be required, including the rate constant for coupling, the concentration of ligand, the nucleophilicity of ligand and quencher, etc.
Further, in biotechnological preparation of target molecules such as proteins, it is well known that to enable an efficient purification thereof, a series of two or more process steps utilising different kinds of separation matrices is often required. U.S. Pat. No. 6,426,315 (Bergström et al.) suggests to replace such a series of steps by using a multifunctional porous separation matrix, i.e. to present the different kind of matrices on a single separation matrix. More specifically, U.S. Pat. No. 6,426,315 relates to a process for preparing such multifunctional porous separation matrices by introducing different functionalities in different layers of the matrix. In brief, the process includes contacting a separation matrix that comprises reactive groups with a reagent, the amount of which is not sufficient for reaction with all groups present in the matrix, and wherein the reaction between the reagent and said reactive groups is rapid compared to the mass transport of the reagent within the matrix. The reactive groups may e.g. be hydroxyl groups, double bonds etc, while the reagent may be a compound that introduces a desired functionality within the matrix, directly or indirectly. In the last mentioned case, the reagent is a compound known as an activating agent, such as a halogenating agent, and the desired functionality is then introduced in a subsequent step. The most preferred functionalities are groups that provide desired separation characteristics to the matrix, commonly known as ligands. Alternatively, the functionalities introduced are the degree of crosslinking, the density or the porosity of the matrix. In order to provide further layers, the reactive groups may be further reacted with another reagent. Thus, the method according to U.S. Pat. No. 6,426,315 may exhibit the drawbacks discussed above under a) and b). In addition, even though the teachings of U.S. Pat. No. 6,426,315 enable the construction of a separation matrix which exhibits a multitude of functions, each one of which will provide different properties as regards binding and diffusion in a separation process, there is no guidance in U.S. Pat. No. 6,426,315 with regard to how to manufacture a separation matrix to that provides an optimal mass transport within the matrix. Thus, there is still a need in this field of alternative methods of producing separation matrices with improved such properties.
In U.S. Pat. No. 5,945,520 (Burton et al.), it is stated that a problem with the known kind of multi or mixed mode chromatography matrices that adsorb a target compound via hydrophobic interactions is that binding efficiencies of less hydrophobic targets will be low unless high salt concentrations are used. To avoid such necessary addition of salt, U.S. Pat. No. 5,945,520 suggests a chromatographic resin, which presents an ionisable ligand comprised of an ionisable functionality and a spacer arm, which attaches said functionality to a solid support matrix. The ionisable functionality is partially electrostatically charged at the pH of adsorption of the target compound to the resin, and is either further charged or of opposite charge at the pH of desorption of the compound from the resin. The ionisable functionalities are selected from a specified group of possible functionalities. In one embodiment, the ionisable functional group is derived from either 2-mercapto-1-methylimidazole or (−)phenylpropanolamine and coupled to a density of at least 150 μmol per milliliter of resin. Such a high ligand density is stated to provide a sufficient hydrophobicity to adsorb target compounds without the need of adding excessive amounts of salt to the liquid. Thus, U.S. Pat. No. 5,945,520 discloses multifunctional ligands evenly coupled to a resin, which consequently can be described as a homogenous separation matrix.
U.S. Pat. No. 6,528,322 (Carlsson et al) relates to an analytical method for qualitative, semi-quantitative or quantitative determination of at least two analytes in an aqueous sample by thin layer chromatography (TLC). More specifically, a method is disclosed wherein analytes, such as isoforms, in an aqueous sample are separated in a flow matrix which permits capillary force assisted fluid flow therethrough, especially a planar flow matrix such as a chromatographic membrane. The gist of the invention is stated to be that the separated analytes are eluted from the separation area of the flow matrix in a direction substantially transverse to the separation direction to migrate to a capture zone. Optionally, the separation zone may have a different ligand density or a gradient of ligand densities along the separation direction. Thus, such a density gradient would be parallel to the flow during use. Furthermore, in this embodiment, there will be a single gradient within each membrane.
Finally, U.S. Pat. No. 5,977,345 relates to an inside-out spatial installation method for a bifunctional reagent that crosslinks a polymer matrix. More specifically, this reference relates to an activated matrix, which can accommodate and optimize the spatial installation of affinity ligands while preventing the immobilization of excess ligand in the outer strata of the hydrogel bead.
However, there is a need in this field of novel separation matrices, which avoid one or more of the problems associated with the prior art. For practical and economical reasons, there is a need in this field of separation matrices that provides an improved mass transport of target molecules.